Biological indicator for monitoring a low-temperature sterilization process

ABSTRACT

An article and method are provided for assessing the efficacy of a sterilization process. The article comprises an outer container having an interior volume; a dry source of measurable biological activity disposed in the interior volume; and an effective amount of a dry agent for neutralizing a sterilant compound; wherein both the source of measurable biological activity and the agent are in vapor communication with an environment outside the outer container. The method comprises exposing the article to a first sterilant and, optionally, a second sterilant for a period of time and detecting the measurable biological activity.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional PatentApplication No. 61/769,357, filed Feb. 26, 2013, which is incorporatedherein by reference in its entirety.

BACKGROUND

Biological indicators (or “sterility indicators”) are devices that areused to test the efficacy of sterilizers, such as those that arecommonly used in hospitals for monitoring processes used to sterilizemedical instruments, glassware, etc. The indicators typically include asource of microorganisms, a culture medium, and a detector to indicatethe presence or absence of viable microorganisms. The culture medium mayalso serve as the detector, with formation of a cloudy suspensionindicating growth of microorganisms. In practice, the source ofmicroorganisms, typically an absorbent paper strip that has beenimpregnated with a predetermined quantity of live microorganisms issubjected to a sterilization process. Thereafter, the microorganismimpregnated strip is placed in a sterile culture medium and incubatedfor a predetermined time at an appropriate temperature. At the end ofthe incubation period, the detector is used to determine whether anymicroorganisms survived the sterilization process. In some indicators,microorganism survival, which means the sterilization was incomplete, isshown by a change in color of the detector.

To simplify the sterilization test process and minimize the risk thatexternal contamination would affect the test results, the elements ofthe biological indicator—microorganisms, culture medium, anddetector—have sometimes been packaged in a way that permits themicroorganism source, culture, and indicator to be combined withoutexposing the biological indicator to the non-sterile surroundings.

Low-temperature sterilization processes are often used to sterilizeobjects that may be damaged by the temperature and/or pressure used insteam sterilization processes. Biological indicators have been developedto monitor the efficacy of low-temperature sterilization processes thatinvolve the use of ethylene oxide, hydrogen peroxide, or peracetic acid,for example. Recently, a sterilization system was developed to permitthe operator to expose an object to two different low-temperaturesterilants. One of the low-temperature sterilants comprises hydrogenperoxide and the other low-temperature sterilant comprises ozone.

SUMMARY

The present disclosure generally relates to biological indicatorarticles and methods to determine the efficacy of a sterilizationprocess. In particular, the present disclosure relates to articles andmethods to determine the efficacy of a sterilization process thatutilizes one or more low-temperature sterilants. Typically, when twolow-temperature sterilants are used in a sterilization process, eachsterilant is used in an amount that is effective to reduce the number ofmicroorganisms present in the sterilizer. Thus, theoretically, if one ofthe sterilant fails to sterilize the object completely (e.g., in alumened portion of the object), the other sterilant should be sufficientto complete the sterilization of the object. The investigators haverecognized the need for an article (e.g., a biological indicator) thatcan be used to distinguish the efficacy of a first quantity of a firststerilant used in a low-temperature sterilization process from theefficacy of second quantity of the first sterilant or a second quantityof a second sterilant that is used in the sterilization processes. Inaddition, the inventive article and method provides a solution to theproblem of distinguishing the efficacy of one of the first quantity fromthe efficacy of the second quantity of the sterilization process.

Advantageously, the article of the present disclosure includes a featurethat, when the article is exposed to a sterilization process utilizing afirst quantity of an otherwise-effective amount of hydrogen peroxide(e.g., hydrogen peroxide vapor) under conditions effective to sterilizean object, protects a measurable biological activity from detrimentaleffects of the first quantity of hydrogen peroxide, even though exposureof the measurable biological activity to the first quantity otherwisewould result in partial or total inactivation of the biologicalactivity.

In one aspect, the present disclosure provides an article. The articlecan comprise an outer container having an interior volume, a first endcomprising an opening, and a second end; a dry source of measurablebiological activity disposed in the interior volume; and an effectiveamount of a dry agent for neutralizing hydrogen-peroxide, the agentbeing disposed in the interior volume. Both the source of measurablebiological activity and the agent are in vapor communication with anenvironment outside the outer container. In any embodiment, the articlefurther can comprise a closure coupled to the outer container, whereinthe closure coupled to the outer container forms a passageway for vaporfrom the environment outside the outer container to pass to the interiorvolume.

In any of the above embodiments, the article further can comprise alayer that is permeable to hydrogen peroxide, wherein the layer iscoupled to the outer container or the closure, wherein the layer isinterposed between the source of measurable biological activity and theenvironment outside the outer container. In any of the aboveembodiments, the layer can be permeable to a second sterilant, whereinthe second low temperature sterilant is a low-temperature sterilantother than hydrogen peroxide. In any of the above embodiments, theopening can comprise a tortuous path. In any of the above embodiments,the source of measurable biological activity can comprise an enzymeactivity or a microorganism capable of reproducing. In any of the aboveembodiments, a portion of the measurable biological activity can bedisposed in a first coating that is optionally disposed on a firstsubstrate. In any of the above embodiments, the agent can be selectedfrom a group consisting of catalase, a thiosulfate salt, a bisulfatesalt, and L-methionine. In any of the above embodiments, a portion ofthe agent can be disposed in the first coating.

In another aspect, the present disclosure provides a method. The methodcan comprise processing an article using a process that comprisesplacing the article in a sterilization chamber, introducing into thechamber a first quantity of a first sterilant, exposing the article tothe first sterilant for a period of time, and detecting a measurablebiological activity. The first quantity of first sterilant can besuspected of comprising an effective amount of hydrogen peroxide. Thearticle can comprise an outer container having an interior volume, afirst end comprising an opening, and a second end; a dry source of themeasurable biological activity disposed in the interior volume; and aneffective amount of a dry agent for neutralizing hydrogen-peroxide, theagent being disposed in the interior volume. Both the source of themeasurable biological activity and the agent are in vapor communicationwith an environment outside the outer container. In any embodiment, themethod further can comprise introducing into the chamber a secondquantity of a second sterilant and exposing the article to the secondsterilant for a period of time.

In any embodiment of the method, the second quantity of second sterilantcan be suspected of comprising hydrogen peroxide, wherein the secondquantity is introduced into the chamber at a predetermined time afterthe first quantity is introduced into the chamber; or the secondquantity of second sterilant can be suspected of comprising an effectiveamount of a sterilant other than hydrogen peroxide. In any of the aboveembodiments of the method, the second sterilant can comprise a gasplasma, ozone, peracetic acid, ethylene oxide or ions derived therefrom.In any of the above embodiments of the method, detecting the measurablebiological activity comprises detecting an enzyme activity. In any ofthe above embodiments of the method, the second sterilant vapor orplasma can comprise ozone, peracetic acid, ethylene oxide or ionsderived therefrom.

In yet another aspect, the present disclosure provides an article. Thearticle can comprise an outer container having an interior volume, afirst end comprising an opening, and a second end; a dry source ofmeasurable biological activity disposed in the interior volume; and aneffective amount of a dry agent for neutralizing a sterilant compound,the effective amount being disposed in the interior volume. Both thesource of measurable biological activity and the agent are in vaporcommunication with an environment outside the outer container. In anyembodiment, the article further can comprise a layer that is permeableto the sterilant compound, wherein the layer is coupled to the outercontainer, wherein the layer is interposed between the source ofmeasurable biological activity and the environment outside the outercontainer. In any embodiment, the source of measurable biologicalactivity can comprise an enzyme activity or a microorganism capable ofreproducing. In any embodiment, the sterilant compound can comprisehydrogen peroxide

The words “preferred” and “preferably” refer to embodiments of theinvention that may afford certain benefits, under certain circumstances.However, other embodiments may also be preferred, under the same orother circumstances. Furthermore, the recitation of one or morepreferred embodiments does not imply that other embodiments are notuseful, and is not intended to exclude other embodiments from the scopeof the invention.

As used herein, “a,” “an,” “the,” “at least one,” and “one or more” areused interchangeably. Thus, for example, a closure comprising “a” windowcan be interpreted to mean that the closure can comprise “one or more”windows.

“Biological indicator” or “BI”, as used herein, refer to an organism(e.g., a microorganism or spore) and/or biological activity (e.g., anenzyme activity) thereof, or an article comprising the organism and/orbiological activity thereof, that is used to monitor the efficacy of asterilization process.

“Self-contained biological indicator” or “SCBI”, as used herein, referto an article containing an organism and/or biological activity suitablefor use as a biological indicator, along with components (e.g.,nutrients, indicator reagents) necessary to detect the inactivation, orlack thereof, of the organism and/or biological activity.

Low-temperature sterilization process”, as used herein, refers to avariety of sterilization processes that do not rely on exposing anobject to be sterilized to steam under pressure (e.g., steam at atemperature greater than about 120 degrees Centigrade) for a period oftime sufficient for the steam to cause sterilization of the object.Nonlimiting examples of low-temperature sterilization processes includeprocesses that rely on exposing the object to compositions of ethyleneoxide, hydrogen peroxide, chlorine dioxide, glutaraldehyde,formaldehyde, or ozone to effect sterilization.

The term “and/or” means one or all of the listed elements or acombination of any two or more of the listed elements.

Also herein, the recitations of numerical ranges by endpoints includeall numbers subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2,2.75, 3, 3.80, 4, 5, etc.).

The features and advantages of the present invention will be understoodupon consideration of the detailed description of the preferredembodiment as well as the appended claims. These and other features andadvantages of the invention may be described below in connection withvarious illustrative embodiments of the invention.

The above summary of the present invention is not intended to describeeach disclosed embodiment or every implementation of the presentinvention. The Figures and the detailed description which follow moreparticularly exemplify illustrative embodiments. Other features, objectsand advantages will become apparent from the description and drawings,and from the claims.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a cross-sectional side view of one embodiment of an articleaccording to the present disclosure.

FIG. 2 is an exploded view the article of FIG. 1.

FIGS. 3A and 3B are exploded side views of two embodiments of asubstrate having a source of measurable biological activity and an agentfor neutralizing hydrogen peroxide disposed thereon.

FIG. 4 is a cross-sectional side view of an embodiment of aself-contained biological indicator having an inner container containinga detection medium according to the present disclosure.

FIG. 5 is an exploded view the article of FIG. 4.

While the above-identified drawing figures set forth several embodimentsof the disclosure, other embodiments are also contemplated, as noted inthe discussion. In all cases, this disclosure presents the invention byway of representation and not limitation. It should be understood thatnumerous other modifications and embodiments can be devised by thoseskilled in the art, which fall within the scope and spirit of theprinciples of the invention. The figures may not be drawn to scale.

DETAILED DESCRIPTION

Before any embodiments of the present disclosure are explained indetail, it is to be understood that the invention is not limited in itsapplication to the details of construction and the arrangement ofcomponents set forth in the following description or illustrated in thefollowing drawings. The invention is capable of other embodiments and ofbeing practiced or of being carried out in various ways. Also, it is tobe understood that the phraseology and terminology used herein is forthe purpose of description and should not be regarded as limiting. Theuse of “including,” “comprising,” or “having” and variations thereofherein is meant to encompass the items listed thereafter and equivalentsthereof as well as additional items. Unless specified or limitedotherwise, the terms “connected” and “coupled” and variations thereofare used broadly and encompass both direct and indirect connections andcouplings. Further, “connected” and “coupled” are not restricted tophysical or mechanical connections or couplings. It is to be understoodthat other embodiments may be utilized and structural or logical changesmay be made without departing from the scope of the present disclosure.Furthermore, terms such as “front,” “rear,” “top,” “bottom,” and thelike are only used to describe elements as they relate to one another,but are in no way meant to recite specific orientations of theapparatus, to indicate or imply necessary or required orientations ofthe apparatus, or to specify how the invention described herein will beused, mounted, displayed, or positioned in use.

The present disclosure generally relates to methods and biologicalindicator articles for determining the efficacy of a sterilizationprocess. In particular, the present disclosure relates to sterilizationprocesses that include exposing an object to hydrogen peroxide (e.g.,hydrogen peroxide vapor and/or plasma). In addition, the presentdisclosure relates to sterilization processes that include exposing anobject to hydrogen peroxide and at least one other low-temperaturesterilant (e.g., gas plasma, ozone, peracetic acid, glutaraldehyde,formaldehyde, ethylene oxide). Gas plasma sterilization ofmicroorganisms is described by Shintani et al (GAS PLASMA STERILIZATIONOF MICROORGANISMS AND MECHANISMS OF ACTION (REVIEW), 2010, Experimentaland Therapeutic Medicine, 1:731-738), which is incorporated herein byreference in its entirety.

Exposing an object to a process that uses two distinct low-temperaturesterilant treatments can provide an additional measure of protection inthe event that one of the low-temperature sterilant treatments fails.The articles of the present disclosure may be used as biologicalindicators or self-contained biological indicators to monitor theefficacy of a sterilization process.

Biological indicators and chemical indicators used to determine theefficacy of a sterilization process are well known in the art. Inconventional biological indicators, a biological activity, such as atest organism which is many times more resistant to the sterilizationprocess employed than most organisms which would be present by naturalcontamination, is coated on a carrier and placed in a sterilizer alongwith the articles to be sterilized. After completion of thesterilization cycle, the carrier is incubated in nutrient medium todetermine whether any of the test organisms survived the sterilizationprocedure. Growth of a detectable number of organisms normally takes aminimum of twenty-four hours. A number of so-called “self-containedbiological indicators” (SCBI) have been described in the patentliterature (e.g., U.S. Pat. Nos. 3,846,242; 4,717,661; 5,073,488;5,223,401; 5,418,167; 5,739,004; 5,795,730; which are all incorporatedherein by reference in their entirety.

In addition, enzyme activities have been used to determine the efficacyof a sterilization process. U.S. Pat. No. 5,073,488; which isincorporated herein by reference in its entirety; describes theusefulness of enzymes (e.g., beta-D-glucosidase, alpha-D-glucosidase,alkaline phosphatase, acid phosphatase, butyrate esterase, caprylateesterase lipase, myristate lipase, leucine aminopeptidase, valineaminopeptidase, chymotrypsin, phosphohydrolase, alpha-D-galactosidase,beta-D-galactosidase, alpha-L-arabinofuranosidase,N-acetyl-8-glucosaminidase, beta-D-cellobiosidase, alanineaminopeptidase, proline aminopeptidase, tyrosine aminopeptidase,phenylalanine aminopeptidase, beta-D-glucuronidase, and a fatty acidesterase) as biological indicators for sterilization processes.

The investigators have found that currently-available biologicalindicators may not be suitable to distinguish the efficacy of anindividual low-temperature sterilant treatment when an object is exposedto a sterilization process that includes exposing the object to aplurality of chemically-distinct, low-temperature sterilant compounds orwhen the object is exposed to a sterilization process that includesdiscrete, sequential exposures to the same sterilant compound (e.g.,hydrogen peroxide). In the situation where the object is exposed to aplurality of chemically-distinct, low-temperature sterilant compounds,this is because both of the two or more chemically-distinct,low-temperature sterilants individually may be capable of inactivatingthe test microorganism and/or enzyme activity of the biologicalindicator. Thus, if exposing the biological indicator to the firststerilant completely inactivates the biological indicator, it is notpossible to determine whether exposure to the second sterilant has anysterilizing effect. In addition to recognizing this need (i.e., for abiological indicator that is capable of determining the efficacy of onlyone sterilant in a process that utilizes at least twochemically-distinct sterilants), the investigators also provide anarticle and method to fulfill the need.

In one aspect, the present disclosure provides an article fordetermining the effectiveness of a sterilization process. Although anarticle made according to the present disclosure can be used to modulatethe resistance of a biological indicator to a sterilization process thatutilizes a single low-temperature sterilant compound, the article isparticularly useful for determining the effectiveness of one sterilantcompound in a low-temperature sterilization process that utilizes twodistinct (e.g., chemically-distinct) low-temperature sterilantcompounds.

The article comprises an outer container having an interior volume, afirst end comprising an opening, and a second end; a dry source ofmeasurable biological activity disposed in the interior volume; and aneffective amount of a dry agent for neutralizing a sterilant compound(e.g., hydrogen peroxide), the effective amount being disposed in theinterior volume. Both the source of measurable biological activity andthe agent are in vapor communication with an environment outside theouter container.

FIG. 1 shows a cross-sectional view and FIG. 2 shows an explodedperspective view of one embodiment of an article 100 according to thepresent disclosure. The article 100 comprises an outer container 10having at least one sidewall 18 defining an interior volume, a first end12 comprising an opening 14, and a second end 16. The opening 14provides fluidic communication (e.g., vapor communication) between theinterior volume of the outer container 10 and an environment (e.g., agaseous environment) external to the outer container 10. In anyembodiment, the opening 14 may comprise a tortuous pathway that isbacteria-impermeable, such as that described in U.S. Pat. No. 4,461,837,incorporated herein by reference, and in commonly assigned copendingU.S. Pat. No. 4,883,641, issued Nov. 28, 1989.

The outer container 10 can have a variety of shapes including, forexample, a right cylindrical shape as shown in FIGS. 1 and 2. The outercontainer 10 can be constructed of a variety of materials including, forexample, glass and plastic. The outer container 10 can be fabricatedusing processes that are known in the art such as, for example,extrusion or molding processes. In any embodiment, the outer container10 can be fabricated using a material that is liquid-impermeable,substantially non-gas absorptive and, optionally, optically transmissive(e.g., translucent or substantially transparent). Non-limiting examplesof suitable materials include, but are not limited to, a variety ofpolyolefins, polyethylene, polypropylene, polycarbonate, polyamide,polymethylpentene, and polyester. In any embodiment wherein the article100 comprises an outer container 10 with an inner container (describedbelow) disposed therein, it is preferable the outer container 10 issufficiently deformable so that a pressure-openable inner container isruptured when the outer container 10 is deformed. In any embodiment, abreaker insert device (not shown) may be contained within outercontainer 10 and which may function to facilitate the breakage of aninner container (described below) when a downward axial force is appliedto the closure 40 of article 100. Such a breaker insert device is taughtin U.S. Patent Application Publication No. 2012/0149094, which isincorporated herein by reference in its entirety.

In any embodiment, the article 100 further comprises an optional closure40 coupled (e.g., by friction fit) to the outer container 10. Theclosure 40 is comprised of a top 41 and depending sidewalls 42. Theclosure 40 restricts vapor communication (e.g., through the opening 14)between the environment and the interior volume of the container 10. Theclosure 40 has a hollow body open at the bottom, with the interiordiameter of the closure 40 being about equal to the exterior diameter ofthe outer container 10, so that closure 40 may be frictionally engagedover the opening 14 of outer container 10. Cut within the sidewalls 42are a plurality of windows 44. The windows 44 facilitate vaporcommunication (e.g., through the opening 14) between the environment andthe interior volume of the container 10. Thus, the closure 40 coupled tothe outer container 10 forms a passageway (e.g., via windows 44) forvapor or plasma from the environment outside the outer container to passto the interior volume of the outer container 10. In any embodiment, theclosure 40 coupled to the outer container 10 can form a passageway thatcomprises a tortuous path (not shown).

Although the illustrated embodiment of FIG. 2 shows windows 44 in thesidewall 42 of the closure 40, it is contemplated that, additionally oralternatively, the top 41 of the closure 40 can have one or more windowextending therethrough (not shown).

When the article 100 is placed in a load to be sterilized, the closure40 is positioned over the opening 14 in the outer container 10 in such amanner that the at least one exterior sidewall 18 of the outer container10 does not block the windows 44. In such a position, sterilant vapor orplasma in the sterilizer may enter outer container 10 by flowing throughwindows 44. Upon completion of the sterilization cycle, the closure 40may be fully engaged with the outer container 10 by depressing it toforce the at least one sidewall 18 of the outer container 10 against theinterior surface of the top 41 of the closure 40, thereby blockingwindows 44. The interior of the outer container 10 is then sealed fromthe outside environment.

In any embodiment, the article 100 preferably further comprises anoptional layer 20. The layer 20 is permeable to hydrogen peroxide vaporand/or hydrogen peroxide plasma. In any embodiment, the layer 20 is alsopermeable to vapor or plasma of at least one other sterilant compoundthat is used in a low-temperature sterilization process. In anyembodiment, the layer 20 may be substantially impermeable tomicroorganisms (e.g., bacteria, spores, yeast, and/or mold).Non-limiting examples of the at least one other sterilant vapor orplasma include a gas plasma (e.g., a plasma of oxygen, nitrogen, helium,or argon), ozone, peracetic acid, and ethylene oxide. In any embodiment,the layer 20 may comprise a nonwoven sheet material such as a spunbondedolefin sheet material, for example.

Other suitable materials that are used to make layer 20 include fibrousmaterials such as cotton, glass wool, cloth, nonwoven webs made frompolypropylene, rayon, polypropylene/rayon, nylon, glass or other fibers,filter papers, microporous hydrophobic and hydrophilic films, opencelled polymeric foams, and semi-permeable plastic films such as thosedescribed in U.S. Pat. No. 3,346,464. Fibrous or cellular materials arepreferred because of the ease with which such materials transmitsterilizing gases. Preferred layer 20 materials include hydrophobicmaterials such as nylon web, microporous hydrophobic film, or glassfiber nonwoven web. Especially preferred is a microporous hydrophobicfilm, commercially available from Celanese Separations Products,Charlotte, N.C., under the trade name “CELGAR K-442 Microporous Film”.

The layer 20 is disposed in the article 100 such that the layer 20 isinterposed between a source of measurable biological activity (describedbelow) and the environment exterior to the outer container 10. Forexample, the layer 20 may be press-fit into the opening 14 of the outercontainer 10 and held in position (e.g., by friction fit) or the layer20 may extend over the opening 14 and held in place by a variety ofmeans including, for example, a clamp, an elastic band, an adhesive, ora closure (discussed below). Alternatively, the layer 20 may bepress-fit into the closure 40, or otherwise coupled to the closure(e.g., via an adhesive or clamp, not shown), and the closure 40 may becoupled to the outer container 10 via friction fit for example, therebypositioning the layer 20 between the interior volume of the outercontainer 10 and the environment external to the outer container asshown in FIGS. 1 and 2

In effect, the fibrous or cellular layer 20 may serve as a filter toprevent the penetration of bacteria and fungi into the outer container10. Hence, layer 20 should have pore sizes no larger than about 0.5microns (e.g., be capable of preventing the passage therethrough ofparticles having dimensions larger than about 0.5 microns). In anyembodiment, the layer may be used in conjunction with the tortuouspathway described above.

The article 100 further comprises a source 30 of measurable biologicalactivity. The source 30 of measurable biological activity is disposed inthe interior volume of the outer container 10. In any embodiment, thesource 30 of biological activity may be disposed in the outer container10 in a position that places at least a portion of the layer 20, ifpresent, between the source 30 of biological activity and theenvironment exterior to the outer container 10. Thus, because theopening 14; the closure 40; if present; and the layer 20, if present;permit the passage of a sterilant compound into the interior volume ofthe outer container 10 according to the present disclosure; the source30 of measurable biological activity disposed in the outer container isin vapor communication with the sterilant compound that is present inthe environment exterior to the outer container 10.

In the absence of a mitigating factor such as an agent for neutralizingthe sterilant compound (e.g., an agent for neutralizing hydrogenperoxide) as described herein, the measurable biological activity in anyarticle of the present disclosure may be susceptible to modulation(e.g., partial or total inactivation) when the source 30 of measurablebiological activity is exposed to an environment comprising thesterilant compound (e.g., hydrogen peroxide vapor or hydrogen peroxideplasma). In any embodiment, in the absence of the mitigating factor, themeasurable biological activity in any article of the present disclosuremay be susceptible to irreversible modulation (e.g., partial or totalinactivation) when the source 30 of measurable biological activity isexposed to an environment comprising a vapor or plasma of the sterilantcompound.

In addition, the measurable biological activity in any article of thepresent disclosure is susceptible to modulation (e.g., partial or totalinactivation) when the source 30 is exposed to an environment comprisingat least one sterilant vapor or plasma (e.g., a gas plasma, ozone,peracetic acid, or ethylene oxide) other than hydrogen peroxide.Preferably, at least a portion the measurable biological activity in anyarticle of the present disclosure is irreversibly modulated (e.g.,partially or totally inactivated) by exposing the source 30 ofmeasurable biological activity to an atmosphere comprising at least onesterilant vapor or plasma other than hydrogen peroxide (e.g., a gasplasma, ozone, peracetic acid, or ethylene oxide). In a more preferredembodiment, exposing any article of the present disclosure thatcomprises a source of measurable biological activity to an efficacioussterilization process that uses a sterilant vapor or plasma (e.g., a gasplasma, ozone, peracetic acid, or ethylene oxide) other than hydrogenperoxide will irreversibly inactivate all of the measurable biologicalactivity.

In any embodiment, the source 30 of measurable biological activity issubstantially dry (e.g., a powder). In any embodiment, at least aportion (e.g., any portion up to and including a 100% portion) of thesource 30 of measurable biological activity may be disposed in a firstcoating (e.g., a dry coating) on a surface. In any embodiment, the firstcoating can be applied to the surface as a liquid coating andsubsequently dried using processes that are known in the art. The source30 of measurable biological activity is disposed in the outer container10 between the opening 14 and the second end 16. Preferably, the source30 of measurable activity is disposed in the outer container 10 betweenthe layer 20, when present, and the second end 16 in a position thatplaces at least a portion of the layer 20 between the source 30 ofbiological activity and the opening 14. In any embodiment, the source 30of biological activity is disposed at a position in the outer container10 that is spaced apart from the opening 14 (e.g., the source ofbiological activity is positioned proximate the second end 16) so that asterilant must penetrate substantially into the outer container 10 tocontact the source 30 of biological activity. Thus, because the layer 20is permeable to a sterilant vapor or plasma (e.g., hydrogen peroxidevapor or plasma), the source 30 of measurable biological activity is invapor communication with sterilant vapor or plasma in the environmentexterior to the outer container 10.

Optionally, the source 30 of measurable biological activity may bedisposed on (e.g., coated on and/or adhered to) a surface of a firstsubstrate 35. The first substrate 35 may take the form of a sheet, forexample. The first substrate 35 is dimensioned to fit inside the outercontainer 10. The first substrate 35 may comprise a metal, glass, glassfibers, a membrane, a nonwoven material, a polymer (e.g., polypropylene,polyethylene, polyester), or a metal-coated polymer, for example. Insome embodiments, the first substrate may comprise a cellulosicmaterial, provided the cellulosic material does not substantially reactwith one or more of the sterilant compounds used in the sterilizationprocess.

In any embodiment, the source 30 of measurable biological activity maycomprise a test microorganism (e.g., a bacterium or a spore) capable ofreproducing. Test microorganisms are conventionally used in a biologicalindicator (hereinafter, “BI”) to monitor sterilization conditions. Theseconventionally-used test microorganisms are generally many times moreresistant to the sterilization process being employed than mostorganisms encountered in natural contamination. The bacterial spore isrecognized as the most resistant form of microbial life. It is the lifeform of choice in all tests for determining the sterilizing efficacy ofsterilization devices, chemicals and processes. Spores from Bacillus andClostridia species are the most commonly-used test microorganisms formonitoring sterilization processes that utilize saturated steam, dryheat, gamma irradiation and ethylene oxide.

In any embodiment, the source 30 of measurable biological activity maycomprise a suspension of bacteria or spores that is coated and driedonto the first substrate 35. In any embodiment, at least a portion(e.g., any portion up to and including a 100% portion) of the source 30of measurable biological activity is disposed on the first substrate 35.Alternatively, the source of measurable biological activity may bedeposited directly into the container (e.g., on a wall 18 of thecontainer proximate the second end, not shown). In these embodiments,the source of measurable biological activity may be deposited as aliquid suspension and subsequently dried using processes that are knownin the art.

The measurable biological activity of a test microorganism (e.g., abacterium or a spore) can be its ability to grow and reproduce. Thus,the biological activity may be detected (e.g., measured) by measuring,either directly or indirectly, a number of microorganisms. In someembodiments, the biological activity may be measured by measuring ametabolic activity (e.g., metabolic by-products such as organic acids,for example) associated with the presence of growing microorganisms. Theproduction of organic acids by growing microorganisms permits thedetection of the biological activity by detecting a drop in pH (e.g., byusing a pH indicator that changes color when the pH drops).

Alternatively, or additionally, the source 30 of measurable biologicalactivity may comprise an enzyme. The enzymes useful in the practice ofthe present invention include extracellular and intracellular enzymes,whose activity correlates with the viability of at least one testmicroorganism commonly used to monitor sterilization efficacy. By“correlates” it is meant that the enzyme activity, over background, canbe used to predict future growth of the test microorganism. The enzymemust be one which, following a sterilization cycle which is sublethal tothe test microorganism, remains sufficiently active to react with asubstrate system for the enzyme, within twenty-four hours, and inpreferred embodiments within eight hours or less, yet be inactivated orappreciably reduced in activity following a sterilization cycle whichwould be lethal to the test microorganism. Exemplary enzymes that can beused according to the present disclosure include alpha-D-glucosidasefrom spores of Geobacillus stearothermophilus, such as thosecommercially available as “ATCC 8005” and “ATCC 7953” from American TypeCulture Collection, Manassas, Va., and beta-D-glucosidase from Bacillusatrophaeus (e.g., commercially available as “ATCC 9372” from AmericanType Culture Collection). Other suitable enzymes and methods foridentifying suitable enzymes can be found in U.S. Pat. No. 5,073,488.

In any embodiment wherein the source of measurable biological activityis an enzyme, the source 30 of measurable biological activity may be: 1)a purified and/or isolated enzyme derived from an appropriatemicroorganism; 2) a microorganism to which the enzyme is indigenous oradded by genetic engineering; or 3) a microorganism to which the enzymehas been added during sporulation or growth, such that the enzyme isincorporated or associated with the microorganism, e.g., an enzyme addedto a spore during sporulation which becomes incorporated within thespore. Preferred microorganisms which may be utilized as a source of anenzyme useful in the practice of the present invention are bacteria orfungi in either the spore or vegetative state. Particularly preferredsources of enzyme include species of microorganisms from the generaBacillus, Geobacillus, Clostridium, Neurospora, and Candida, forexample.

Returning to the drawings, the article further comprises an agent 50 forneutralizing a sterilant compound (e.g., hydrogen peroxide). The agent50 is disposed in the interior of the article (e.g., the interior volumeof the outer container 10). Hydrogen peroxide is typically neutralizedby decomposing it to water and oxygen. In this specification and theappended claims, with respect to hydrogen peroxide, “neutralize” and“decompose” are used interchangeably to refer to the process by whichthe hydrogen peroxide is inactivated. Suitable agents for neutralizinghydrogen peroxide include a variety of compositions including, forexample, catalase, peroxidase, and other peroxide-neutralizingcatalysts. A preferred composition is catalase, more preferablyfreeze-dried catalase powder. Suitable agents for neutralizing ozoneinclude, for example, thiosulfate, metal ions (Cu+), and nitryl ions.Suitable agents for neutralizing formaldehyde include, for example,glycine. Suitable agents for neutralizing glutaraldehyde include forexample, a bisulfite compound (see, for example, Susan L. P. Jordan(1996): INACTIVATION OF GLUTARALDEHYDE BY REACTION WITH SODIUMBISULFITE, Journal of Toxicology and Environmental Health, 47:3,299-309); and H-W Leung (2001) ECOTOXICOLOGY OF GLUTARALDEHYDE: REVIEWOF ENVIRONMENTAL FATE AND EFFECTS STUDIES, Ecotoxicology andEnvironmental Safety, 49:26-39; which are both incorporated herein byreference in their entirety).

In any embodiment, the agent for neutralizing a sterilant compound maycomprise a dry composition (e.g., a dry mixture) comprising a pluralityof compounds (e.g., sodium bisulfite and sodium thiosulfate). Theplurality of compounds may neutralize the same sterilant compound(s)and/or they may neutralize different sterilant compound(s). The use ofliquid mixtures of compounds to neutralize high-level disinfectants isdescribed, for example, in Espigares et al. (EFFICACY OF SOMENEUTRALIZERS IN SUSPENSION TESTS DETERMINING THE ACTIVITY OFDISINFECTANTS, J. Hospital Infection, 2003, 55:137-140), which isincorporated herein by reference in its entirety.

The agent 50 for neutralizing a sterilant compound optionally is mixedwith a stabilizer (e.g., a powder), such as a sugar, a salt, otherstabilizers well known in the art, or combinations thereof. Thestabilizer not only extends the shelf life of the catalyst but alsoincreases the quantity of powder to be used. This is desirable, sincevery little pure agent (e.g., and enzyme such as catalase) would beneeded for an article of the present disclosure. Preferably, thestabilizer does not interfere with growth of a microorganism, ifpresent, in the article 100. Other suitable agents for neutralizinghydrogen peroxide include a thiosulfate salt, a bisulfate salt, andL-methionine.

Metal catalysts, such as platinum, palladium, iron, etc, are alsosuitable hydrogen peroxide neutralizers. Preferably, the metal catalystdoesn't inhibit the growth of a microorganism, if present, in thearticle. Because high surface area may be desirable to facilitate rapidneutralization of hydrogen peroxide, the preferred form of a metalcatalyst is either a fine powder of the metal or a coating of the metalon a fine ceramic powder.

The amount of agent 50 disposed in the container 10 should be sufficientto neutralize enough sterilant (e.g., hydrogen peroxide) to protect(i.e., prevent the killing or inactivation of) at least a portion of themeasurable activity while the article 100 is exposed to typicalsterilization process that utilizes an environment having enough of thesterilant (e.g., hydrogen peroxide) vapor or plasma to otherwise kill orinactivate all of the biological activity. The amount of agent used toneutralize hydrogen peroxide sterilant may vary depending upon whetherthe agent reacts with the hydrogen peroxide catalytically (i.e., onemolecule of agent can react with more than one molecule of hydrogenperoxide) or stoichiometrically (i.e., one molecule of agent can reactwith only one molecule of hydrogen peroxide). The amount of neutralizingagent used in an article may also depend upon the hydrogen peroxidesterilization process in which it is used. The Examples herein provideguidance as to how much agent can be used and provide a simple procedurethat can be used to test any given agent with any given sterilant (e.g.,hydrogen peroxide) that is used in a low-temperature sterilizationprocess.

In any embodiment, the agent 50 for neutralizing a sterilant compound issubstantially dry (e.g., a powder). In any embodiment, at least aportion (e.g., any portion up to and including a 100% portion) of theagent 50 may be disposed in a second coating (e.g., a dry coating) on asurface. In any embodiment, the second coating can be applied to thesurface as a liquid coating and subsequently dried using processes thatare known in the art. The agent 50 is disposed in the outer container 10between the layer 20 and the second end 16. The agent 50 is disposed inthe outer container 10 in a position that places at least a portion ofthe layer 20 between the agent 50 and the opening 14. Thus, because thelayer 20 is permeable to sterilant (e.g., hydrogen peroxide) vapor orplasma, the agent 50 is in vapor communication with sterilant vapor orplasma in the environment exterior to the outer container 10.

The agent 50 may be disposed in a variety of locations in the outercontainer 10. In any embodiment, at least a portion (e.g., any portionup to and including a 100% portion) of the agent 50 may be disposed onthe first substrate 35, as shown in FIGS. 3A and 3B. Alternatively, theagent may be deposited directly into the container (e.g., on a wall ofthe container proximate the second end, not shown). In any embodiment,the agent may be deposited as a liquid suspension and subsequently driedusing processes that are known in the art. Preferably, the agent 50 isdisposed in the outer container 10 proximate the source 30 of measurablebiological activity.

In any embodiment, the agent 50 is disposed in contact with the source30 of measurable biological activity. For example, the source 30 ofmeasurable biological activity may be disposed as a first coating on afirst substrate 35 and the agent 50 may be disposed as a second coatingthat overlaps at least a portion of the source 30, as shown in FIG. 3A.Alternatively, the source 30 of measurable biological activity may bedisposed as a first coating on a first substrate 35 and the agent 50 maybe disposed as a second coating adjacent the first coating, as shown inFIG. 3A. In an alternative embodiment (not shown), the source ofmeasurable biological activity is disposed as a first coating on a firstsubstrate and the agent for neutralizing hydrogen peroxide is disposedas a second coating on a second substrate. In any embodiment, the secondsubstrate may comprise the layer 20 described herein. In theseembodiments, the second coating can be applied to a surface of the layer20 facing the interior volume of the outer container 10. Thus, in anyembodiment, the agent 50 for neutralizing a sterilant may be disposedproximate the opening 14 and the closure (discussed below), if present.

In any embodiment, the article further may comprise a nutrient (notshown) to facilitate the germination or outgrowth of a spore. In anyembodiment, the article further may comprise an indicator (not shown) toindicate growth of a microorganism and/or indicate a presence ofmeasurable enzyme activity. The nutrient and/or indicator may bedisposed in the first coating or second coating described hereinabove.

In any embodiment, an article of the present disclosure may furthercomprise an inner container disposed in the outer container. FIGS. 4 and5 show two views of one embodiment of an article 200 comprising an innercontainer according to the present disclosure. The article 200 comprisesan outer container 10, layer 20, and closure 40, as describedhereinabove. The article 200 also comprises a first substrate 35 onwhich the source of measurable biological activity (not shown) and agentfor neutralizing a low-temperature sterilant (e.g., hydrogen peroxide),both described hereinabove, are disposed. In addition, the article 200comprises an inner container 60 disposed in the outer container 10. Theinner container 60 is disposed in the outer container 10 between thelayer 20 and the second end 16. The inner container 60 contains areagent 65 to facilitate the detection of the biological activity. Inany embodiment, the reagent can be dissolved or suspended in an aqueousliquid.

The inner container 60 may contain an aqueous solution of an enzymesubstrate and/or an aqueous nutrient medium. The inner container 60 isfabricated using material which is impermeable to gases and liquids andis capable of being opened upon the application of pressure thereto(i.e., “pressure openable”) to permit the enzyme substrate and/ornutrient medium to enter the outer container 10. The inner container 60is preferably of frangible material, such as glass; and is preferablysnugly carried within the outer container 10 in coacting relationshiptherewith to permit breakage or crushing of the inner container 60 whenthe outer container 10 is deformed. In another embodiment (not shown),the inner container may be sealed with a plug such that the plug isexpelled to release the contents of the inner container upon applicationof pressure. In still another embodiment (not shown), the closure mayinclude an ampoule crushing device, as shown in U.S. Pat. No. 4,304,869,wherein the closure has tabs depending from the bottom of the closuredevice which, upon depression of the closure device, serve to crush theampoule.

In an embodiment wherein the source of measurable biological activity isa microorganism and detection of the biological activity comprisesdetecting growth of the microorganism, the reagent may comprise anutrient to facilitate the germination of a microbial spore and/orfacilitate the growth of the microorganism. In an embodiment wherein thesource of measurable biological activity is a microorganism (e.g., amicroorganism from a germinated spore) and detection of the biologicalactivity comprises detecting growth of the microorganism, the reagentmay comprise an indicator (e.g., a pH indicator, a chromogenic enzymesubstrate, a fluorogenic enzyme substrate) to facilitate the detectionof a viable microorganism. Suitable reagents (i.e., nutrients, nutrientmedia, growth media, and indicators) for use in detecting a source ofmeasurable biological activity comprising a microorganism are described,for example, in U.S. Pat. Nos. 3,346,464; 3,585,112; 4,291,122;4,416,984; 4,528,268; 5,073,488; and 5,223,401; which are allincorporated herein by reference in their entirety.

In an embodiment wherein the source of measurable biological activity isan enzyme activity and detection of the biological activity comprisesdetecting the enzyme activity, the reagent of the present disclosure maycomprise an enzyme substrate. Suitable indicators for detecting a sourceof measurable biological activity comprising an enzyme activity include,for example, a redox indicator, a chromogenic enzyme substrate, and afluorogenic enzyme substrate. Non-limiting examples of suitableindicator reagents to detect an enzyme activity according to the presentdisclosure are disclosed in U.S. Pat. Nos. 5,073,488 and 6,355,448;which are incorporated herein by reference in their entirety.

In another aspect, the present disclosure provides a method fordetermining the effectiveness of a sterilization process. The methodcomprises processing the article of any one of the embodiments of thepresent disclosure using a sterilization process and detecting themeasurable biological activity of the article. The article comprises adry source of measurable biological activity and an effective amount ofa dry agent for neutralizing a low-temperature sterilant compound (e.g.,hydrogen peroxide), as described herein. The sterilization processcomprises placing the article in a sterilization chamber; introducinginto the chamber a first quantity of a first sterilant, wherein thefirst quantity of first sterilant is suspected of comprising aneffective amount of hydrogen peroxide; and exposing the article to thefirst sterilant for a period of time.

The sterilization chamber can be any chamber suitable for conducting alow-temperature sterilization process. Non-limiting examples of suitablesterilization chambers have been described for low-temperaturesterilization processes using ethylene oxide (see, for example, U.S.Pat. No. 4,337,223), hydrogen peroxide (see, for example, U.S. Pat. No.6,953,549, which is incorporated herein by reference in its entirety),and ozone (see, for example, U.S. Patent Application Publication No. US2011/0076192, which is incorporated herein by reference in itsentirety).

Typically, processing an article in a sterilization processes comprisesplacing an article to be sterilized into a vessel and either introducinginto the vessel or generating in the vessel an effective amount of thesterilant vapor or plasma (e.g., the first sterilant vapor or plasma orsecond sterilant vapor or plasma), thereby establishing in the vessel anenvironment (e.g., a gaseous and/or plasma environment) to which thearticle is exposed. Preferably, the article is held in the vessel underconditions suitable for an effective amount of the first sterilant vaporor plasma to sterilize the article. A person having ordinary skill inthe art will recognize the suitable conditions. Suitable conditions mayinclude, but are not limited to, the amount and type of material loadedinto the vessel, the temperature of the vessel, the atmospheric pressurein the vessel, the relative humidity of the vessel, the length of timethe article is held in the vessel, and a combination of two or more ofthe foregoing conditions.

The first sterilant can be any sterilant compound that is suitable foruse in a low-temperature sterilization process as a vapor and/or plasma;the sterilant compound being a compound for which a correspondingneutralizer, which can be provided in the article in a substantially dryform, is identified. Nonlimiting examples of suitable first sterilantcompounds include hydrogen peroxide and ozone.

The method comprises introducing into the sterilization chamber a firstquantity of the first sterilant. Typically, the first quantityintroduced into the chamber is a quantity suspected of being aneffective amount of sterilant. An “effective amount” of sterilant, asused herein, refers to a quantity of sterilant compound introduced intoa sterilization chamber (i.e., the sterilant is introduced into thechamber as a vapor-phase or plasma-phase and/or the sterilant isintroduced into the chamber (e.g., as a liquid or solid) and isconverted in the chamber into a vapor-phase and/or plasma-phase). Thefirst quantity of the first sterilant is sufficient, according totypical parameters (e.g., temperature, exposure time, relative humidityor a combination of any two of the foregoing parameters) for using thefirst sterilant in a low-temperature sterilization process to reduce thenumber of viable test microorganisms (e.g., spores that are susceptibleto the sterilant compound) in a biological indicator by at least atwo-log reduction.

In any embodiment, the first sterilant comprises hydrogen peroxide.Sterilization processes that comprise a step of exposing an article toan environment comprising an effective amount of vapors or plasma ofhydrogen peroxide are known in the art and are described, for example,in U.S. Pat. Nos. 4,169,123; 4,169,124; 4,643,876; and 4,756,882; whichare all incorporated herein by reference in their entirety. In addition,commercial sterilizers (e.g., a STERRAD NX sterilizer, available fromAdvanced Sterilization Products (ASP) of Irvine, Calif.; an AMSCO V-PRO1 sterilizer, available from STERIS Corporation of Mentor, Ohio) includesterilization vessels and automated programs to expose an article to anenvironment comprising an effective amount of vapors or plasma ofhydrogen peroxide.

Some of the automated programs for hydrogen peroxide sterilizationprocesses provide the capability of introducing (e.g., in a generallycontinuous manner) hydrogen peroxide vapor (VHP) into the sterilizationchamber until the concentration of the VHP reaches a predeterminedvalue. The amount of VHP in the chamber can be estimated by measuringthe pressure in the chamber as the VHP is introduced. Other automatedprograms provide the capability of introducing at least two discretequantities of hydrogen peroxide at separate times into the chamber.Thus, in any embodiment, the method further can comprise introducinginto the chamber a second quantity of a second sterilant, wherein thesecond sterilant comprises hydrogen peroxide. The first introduction ofhydrogen peroxide introduces an effective amount into the chamber and asubsequent injection of hydrogen peroxide replenishes the vapor hydrogenperoxide that may have depleted due to condensation, degradation, and/orinteraction with materials (including microorganisms) present in theload that is being treated and, thus, ensuring the efficacy of thesterilization process. In any embodiment, the second quantity can besuspected of comprising an effective amount of hydrogen peroxide.

Processing the article according to the method comprises exposing thearticle to the first sterilant for a period of time. A person havingordinary skill in the art will recognize, in order to decontaminate orsterilize an object (e.g., the article of the present disclosure), theefficacy of the process depends on the quantity of sterilant (e.g., theconcentration of sterilant in the sterilization chamber), the length oftime to which the object is exposed to the sterilant, and may dependupon other factors such as, for example, the temperature of the loadand/or the chamber, relative humidity, and the amount and/or type (e.g.absorbent property, relative to the sterilant) of material present inthe sterilization chamber. The Examples below provide general guidancefor a typical process; however the parameters can be varied according tothe desired result, as is well known by a person having ordinary skillin the art.

As discussed above, in any embodiment, a method of the presentdisclosure comprises introducing into the chamber a second quantity of asecond sterilant. The second sterilant can be introduced into thechamber as a vapor or plasma or, alternatively, can be transformed inthe chamber into a vapor or plasma form. In any embodiment, the secondsterilant comprises a sterilant compound other than hydrogen peroxide.Suitable second sterilants include, but are not limited to, a gasplasma, ozone, chlorine dioxide, peracetic acid, ethylene oxide or ionsderived therefrom. In any embodiment of the method, the second quantitycan be suspected of comprising an effective amount of second sterilant.

Sterilization processes that comprise a step of exposing an article toan environment comprising an effective amount of ozone are known in theart and are described, for example, in PCT Patent Publication No. WO92/04057, which is incorporated herein by reference in its entirety.Sterilization processes that comprise a step of exposing an article toan environment comprising an effective amount of vapor-phase peraceticacid also are known in the art and are described, for example, in PCTPatent Publication No. WO 2012/173756, which is incorporated herein byreference in its entirety. Sterilization processes that comprise a stepof exposing an article to an environment comprising an effective amountof gaseous ethylene oxide also are known in the art (see, for example,U.S. Pat. No. 4,337,223, and U.S. Patent Application Publication No. US2007/0292305, which are incorporated herein by reference in theirentirety).

Recently, sterilizers that are capable exposing an article to twodifferent types of sterilant vapors and/or plasmas have been developed.An exemplary sterilizer with the capability of exposing an article totwo distinct sterilant compositions is described in U.S. PatentApplication Publication No. 2011/0076192, which is incorporated hereinby reference in its entirety. A commercial sterilizer (3M OPTREOZ 125-ZSterilizer, or its equivalent, available from TS03 of Quebec City,Canada) is capable of introducing into a sterilization chamber a firstquantity of a first sterilant (e.g., vapor-phase hydrogen peroxide) anda second quantity of a second sterilant (e.g., ozone) in order tosterilize the article. Thus, processing an article using a sterilizationprocess according to the present disclosure can comprise placing thearticle into a sterilization vessel (e.g., a sterilization vesselsimilar to the 3M OPTREOZ 125-Z Sterilizer) that is adapted to exposethe article to two distinct sterilants.

According to any embodiment of the method of the present disclosure,after the article has been processed (e.g., exposed to the firststerilant and/or second sterilant for a period of time), the measurableactivity is detected. The procedure used to detect the measurableactivity will vary according to the nature of the source of measurablebiological activity and the components present in the article.

For example, when the source of measurable biological activity comprisesa viable microorganism (e.g., a bacterium, a yeast, or a spore),detecting the measurable biological activity can comprise using aculture medium to culture the microorganism. In these embodiments,detecting the measurable biological activity may comprise incubating thesource of biological activity in contact with the culture medium for aperiod of time. A person having ordinary skill in the art will recognizethe incubation temperature is selected according to the typicalrequirements of the particular microorganism being cultured. Forexample, a spore of Bacillus atrophaeus may be incubated at atemperature around 37 degrees C., whereas a spore of Geobacillusstearothermophilus may be incubated at a temperature around 56 degreesC.

The microorganism can be cultured in the article (e.g., by adding asuitable culture medium to the article after the sterilization processor by rupturing the inner container to release into the outer containera suitable culture medium and/or indicator reagent contained in theinner container). A person having ordinary skill in the art willrecognize a variety of methods to detect a plurality of microorganismsin a culture medium. The methods include, but are not limited to, usinga chromogenic or fluorogenic enzyme substrate to observe an enzymaticreaction associated with the viable microorganism, using a chromogenicor fluorogenic redox indicator to observe and oxidation or reductionreaction associated with the viable microorganism, using a pH indicatorto observe the production of an acidic or basic metabolite produced by aviable microorganism.

The types of nutrient media usefully employed in the present inventionare widely known in the art. Examples of preferred nutrient media areaqueous solutions of soybean-casein digest broth, fluid thioglycollateand Dextrose Tryptone (Difco Laboratories, Inc.) A modified tryptic soybroth base, without glucose, is especially preferred. To avoidcontamination, such aqueous nutrient media normally is sterilized afterhaving been placed in the inner container. Commonly known microbialgrowth indicators, which change color in the presence of viablemicroorganisms, are preferably present in at least one of thecontainers. The growth indicator material preferably is soluble in, andimparts color (upon microorganism growth) to, the aqueous nutrientmedium so that a change in color may be easily observed through thetranslucent walls of the outer container. In addition, the growthindicator material is preferably selected so that it will not interferewith the color or luminescence of the enzyme-modified product. Growthindicator materials which may be employed in the present invention arewell known to the art and include pH-sensitive dye indicators (such asbromothymol blue, bromocresol purple, phenol red, etc.),oxidation-reduction dye indicators (such as methylene blue, etc.). Suchmaterials commonly undergo changes in color in response to a phenomenonof microorganism growth, such as changes in pH, oxidation-reductionpotentials, etc.

Observing a reaction associated with a viable microorganism can compriseusing an instrument to detect the reaction. Observing a reactionassociated with a viable microorganism can comprise using an instrumentto measure a product (e.g., a chromogenic or fluorogenic product) of thereaction. In any embodiment, detecting the measurable biologicalactivity may comprise detecting the absence of the measurable biologicalactivity.

When the source of measurable biological activity comprises an enzymeactivity, detecting the measurable biological activity can compriseusing an enzyme substrate system (e.g., comprising a luminescent enzymesubstrate, a chromogenic enzyme substrate or a fluorogenic enzymesubstrate) to detect the measurable biological activity. In theseembodiments, detecting the measurable biological activity may compriseincubating the source of biological activity in contact with the enzymesubstrate for a period of time.

In the context of this application, an enzyme substrate system is bydefinition a substance or mixture of substances acted upon by an enzymeand converted into an enzyme-modified product. In general, the enzymemodified product is a luminescent, fluorescent, colored or radioactivematerial. However, the enzyme substrate system can consist of a compoundwhich when reacted with the enzyme, will yield a product which willreact with an additional compound or composition to yield a luminescent,fluorescent, colored or radioactive material. Preferably, where thesubstrate system is to be included in the indicator device duringsterilization, the substrate must not spontaneously break down orconvert to a detectable product during sterilization or incubation. Forexample, in devices used to monitor a process using hydrogen peroxidevapors or plasma and ozone vapors or plasma, the components of thesubstrate system should be stable at temperatures between about 20° and80° C. Preferably also, where the enzyme substrate system is to bestored in and/or used with conventional growth media, it should bestable (e.g., not substantially spontaneously hydrolyze) when in contactwith the growth media.

Detecting a measurable biological activity that comprises an enzymeactivity comprises observing a product of the reaction catalyzed (e.g.,directly or indirectly) by the enzyme activity. Observing a product of areaction catalyzed by the enzyme activity can comprise using aninstrument to detect the product. Observing a product of a reactioncatalyzed by the enzyme activity can comprise using an instrument tomeasure the product (e.g., light, a colored product, or fluorescentproduct). In any embodiment, detecting the measurable biologicalactivity may comprise detecting the absence of the measurable biologicalactivity.

A preferred method of monitoring a fluorescent product of a measurablebiological activity of the present disclosure is to use a fluorimeterdesigned specifically (e.g., configured to be operably coupled to theouter container) for the articles of the present disclosure. Afluorimeter eliminates the subjective interpretation encountered whenattempting to visually differentiate between low levels of a fluorescentproduct and background fluorescence (e.g. due to components of anutrient medium, if present) or no fluorescence. A fluorimeter can becalibrated to detect a minimum amount of fluorescent product within agiven incubation period.

A particularly preferred fluorimeter, designed for use with the devicesof this invention, consists of a chamber designed to block ambient lightwhile positioning the outer container of the article such that thesource of measurable biological activity within the outer container canbe illuminated with a 365 nm wavelength ultraviolet light, and aphotodiode can be used detect any resultant fluorescence in the 460 nmwavelength region. The fluorimeter may be calibrated to detect athreshold amount of fluorescence to distinguish the presence ofmeasurable biological activity.

The method of the present disclosure can be used to determine theeffectiveness of a quantity of second sterilant used in a sterilizationprocess that utilizes first and second sterilant compounds as describedherein. Advantageously, this is possible whether the first and secondsterilant compounds are the same compound or are different compounds.The agent for neutralizing a sterilant compound (e.g., a first sterilantcompound such as hydrogen peroxide) prevents the first sterilantcompound from inactivating (e.g., partially or totally inactivating) thesource of measurable biological activity. Therefore, if the measurableactivity is detected (e.g., at least a portion or all of the measurableactivity is detected) after the article is exposed to both the first andsecond sterilant compounds, it can be concluded that the sterilizationprocess, in general, and the quantity of second sterilant compound, inparticular, were not effective to sterilize the article.

Exemplary Embodiments

Embodiment A is an article, comprising:

an outer container having an interior volume, a first end comprising anopening, and a second end;

a dry source of measurable biological activity disposed in the interiorvolume; and

an effective amount of a dry agent for neutralizing hydrogen peroxide,the agent being disposed in the interior volume;

wherein both the source of measurable biological activity and the agentare in vapor communication with an environment outside the outercontainer.

Embodiment B is the article of Embodiment A, further comprising aclosure coupled to the outer container, wherein the closure coupled tothe outer container forms a passageway for vapor or plasma from theenvironment outside the outer container to pass to the interior volume.

Embodiment C is the article of Embodiment A or Embodiment B, furthercomprising a layer that is permeable to hydrogen peroxide, wherein thelayer is coupled to the outer container or the closure, wherein thelayer is interposed between the source of measurable biological activityand the environment outside the outer container.

Embodiment D is the article of Embodiment C, wherein the layer ispermeable to hydrogen peroxide and a second low-temperature sterilant,wherein the second low temperature sterilant is a low-temperaturesterilant other than hydrogen peroxide.

Embodiment E is the article of any one of the preceding claims, whereinthe opening or the passageway comprises a tortuous path.

Embodiment F is the article of any one of the preceding embodiments,wherein the measurable biological activity comprises an enzyme activity.

Embodiment G is article of any one of Embodiments A through E, whereinthe measurable biological activity is a microorganism capable ofreproducing.

Embodiment H is the article of Embodiment G, wherein the microorganismis an endospore.

Embodiment I is the article of any one of the preceding Embodiments,wherein the second sterilant is selected from a group consisting of agas plasma, ozone, peracetic acid, and ethylene oxide.

Embodiment J is the article of any one of the preceding Embodiments,wherein a portion of the source of measurable biological activity isdisposed in a first coating.

Embodiment K is the article of Embodiment J, wherein the article furthercomprises a first substrate, wherein the first coating is disposed onthe first substrate.

Embodiment L is the article of any one of the preceding Embodiments,wherein the agent is selected from a group consisting of catalase, athiosulfate salt, a bisulfate salt, and L-methionine.

Embodiment M is the article of any one of the preceding Embodiments,wherein a portion of the agent is disposed in the first coating.

Embodiment N is the article of any one of the preceding Embodimentswherein a portion of the agent is disposed in a second coating.

Embodiment O is the article of Embodiment N, wherein the article furthercomprises a second substrate, wherein a portion of the second coating isdisposed on the second substrate.

Embodiment P is the article of Embodiment O, wherein the layer is thesecond substrate, wherein the second coating is disposed on or in thesecond substrate.

Embodiment Q is the article of any one of Embodiments N through P,wherein the second coating is proximate the first coating.

Embodiment R is the article of Embodiment Q, wherein a portion of thesecond coating overlays the first coating.

Embodiment S is the article of any one of Embodiments G through R,further comprising a nutrient to facilitate growth of the microorganism.

Embodiment T is the article of any one of the preceding Embodiments,further comprising an indicator capable of reacting with the source ofmeasurable biological activity.

Embodiment U is the article of Embodiment S or Embodiment T, wherein thenutrient or the indicator is disposed in an inner container that isdisposed in the outer container between the first end and the secondend.

Embodiment V is a method, comprising:

processing the article of any one of the preceding Embodiments using aprocess that comprises:

-   -   placing the article in a sterilization chamber;    -   introducing into the chamber a first quantity of a first        sterilant, wherein the first quantity of first sterilant is        suspected of comprising an effective amount of hydrogen        peroxide;    -   exposing the article to the first second sterilant for a period        of time; and

detecting the measurable biological activity.

Embodiment W is the method of Embodiment V, further comprising:

introducing into the chamber a second quantity of a second sterilant;and

exposing the article to the second sterilant for a period of time.

Embodiment X is the method of Embodiment W, wherein the second quantityof second sterilant is suspected of comprising hydrogen peroxide,wherein the second quantity is introduced into the chamber at apredetermined time after the first quantity is introduced into thechamber.

Embodiment Y is the method of Embodiment W, wherein the second quantityof second sterilant is suspected of comprising an effective amount of asterilant other than hydrogen peroxide.

Embodiment Z is the method of Embodiment X, wherein the second quantityis introduced into the chamber after the first quantity is introducedinto the chamber.

Embodiment AA is the method of any one of Embodiments V through Z,wherein exposing the article to a first sterilant comprises exposing thearticle to the first sterilant under conditions suitable for efficacy ofthe first sterilant.

Embodiment BB is the method of any one of Embodiments V through AA,wherein exposing the article to a second sterilant comprises exposingthe article to the second sterilant under conditions suitable forefficacy of the second sterilant.

Embodiment CC is the method of any one of Embodiments W through BB,wherein the second sterilant comprises a gas plasma, ozone, peraceticacid, ethylene oxide or ions derived therefrom.

Embodiment DD is the method of any one of Embodiments V through CC,wherein detecting the measurable biological activity comprises detectingan enzyme activity.

Embodiment EE is the method of any one of Embodiments V through DD,wherein detecting the measurable biological activity comprises detectinga pH change.

Embodiment FF is an article, comprising:

an outer container having an interior volume, a first end comprising anopening, and a second end;

a dry source of measurable biological activity disposed in the interiorvolume; and

an effective amount of a dry agent for neutralizing a low-temperaturesterilant compound, the effective amount being disposed in the interiorvolume;

wherein both the source of measurable biological activity and the agentare in vapor communication with an environment outside the outercontainer.

Embodiment GG is the article of Embodiment FF, further comprising alayer that is permeable to the low-temperature sterilant compound,wherein the layer is coupled to the outer container, wherein the layeris interposed between the source of measurable biological activity andthe environment outside the outer container.

Embodiment HH is the article of Embodiment FF, wherein the openingcomprises a tortuous path.

Embodiment II is the article of any one of Embodiments FF through HH,wherein the source of measurable biological activity comprises an enzymeactivity.

Embodiment JJ is the article of any one of Embodiments FF through HH,wherein the source of measurable biological activity is a microorganismcapable of reproducing.

Embodiment KK is the article of any one of Embodiments FF through JJ,wherein the low-temperature sterilant compound comprises hydrogenperoxide.

Advantages and embodiments of this disclosure are further illustrated bythe following examples, but the particular materials and amounts thereofrecited in these examples, as well as other conditions and details,should not be construed to unduly limit this disclosure. All materialsare commercially available or known to those skilled in the art unlessotherwise stated or apparent.

EXAMPLES

Sterilizer Systems and Sterilization Cycle Parameters.

The biological indicators (BIs) described in the Examples below, wereexposed to sterilization cycles using hydrogen peroxide sterilizationwith a STERRAD NX sterilizer (available from Advanced SterilizationProducts (ASP) of Irvine, Calif.) and the 3M OPTREOZ 125-Z Sterilizer(available from T503 of Quebec City, Canada). The sterilantconcentration and exposure time was varied. In the STERRAD NXsterilizer, the BIs were exposed to a standard (“full”) cycle and a 3minute (“half”) cycle exposure. The half cycle used a smaller quantityof the sterilant, hydrogen peroxide, injected into the chamber. In the3M OPTREOZ 125-Z sterilizer, some of the BIs were exposed to a full“Cycle 2” which used hydrogen peroxide followed by ozone, and some ofthe BIs were exposed to a 11.84 Torr (1.58 kPa) hydrogen peroxide-onlycycle. The descriptions of the STERRAD NX cycles and OPTREOZ cycles arelisted in Tables 1 and 2 below.

TABLE 1 STERRAD NX sterilizer: Standard cycle and 3 minute exposurecycles Cycle stages Controlled parameter Standard Cycle 3 min HalfCycle 1) Delivery Volume of 59% H₂O₂ 1.8 mL 1.0 mL solution admittedinto the sterilizer 2) Exposure Exposure to H₂O₂   3 minutes   3 minutesNumber of phases 2 1 Approx. cycle duration  28 minutes  14 minutes(empty chamber)

TABLE 2 OPTREOZ 125 Z sterilizer: Cycle 2 and hydrogen peroxide onlycycles Hydrogen Peroxide Cycle stages Controlled parameter Cycle 2only 1) Vacuum Pressure 1 Torr 1 Torr (0.133 kPa) (0.133 kPa) 2) H₂O₂Injection P_(v) of 50% H₂O₂ 19 Torr 11.84 Torr solution admitted (2.53kPa) (1.58 kPa) 3) O₃ Injection Ozone dose injected 10 mg/L 0 mg/L 4)Exposure Duration 5 minutes 0 minutes Number of phases 2 1 Approx. cycleduration (empty chamber) 56 minutes Less than 50 minutes

Examples 1-5

The effect of catalase, as an agent for neutralizing hydrogen peroxide,on protecting spores from exposure to a hydrogen peroxide sterilizationprocess was evaluated by positioning catalase at different locationsinside example biological indicators. Lyophilized catalase powder frombovine liver, available from Sigma Aldrich, St. Louis, Mo., was used inthe examples described below. Commercially available 3M ATTEST 1274Biological Indicators and 3M ATTEST 1292 Rapid Readout BiologicalIndicators, were modified by the addition of catalase as follows: Tensample biological indicators (BIs) were prepared for each of Examples1-4 and 12 samples were prepared for Example 5. After the Examples 1-5were prepared as described below, they were subjected to the STERRAD NXsterilizer standard cycle, described in Table 1, above. Table 3 belowshows the results of the sterilized biological indicators as a ratio ofpositive/total BIs evaluated. A positive result implies that thecatalase enzyme activity was effective in preventing the hydrogenperoxide sterilant from killing all the spores in the BI.

The Biological Indicator article used in Example 1 was prepared by firstdisassembling several 3M ATTEST 1274 Biological Indicators, availablefrom 3M Company of St. Paul, Minn. The component parts of the 1274Biological Indicators included an outer container (sleeve) with anopening, a sealed glass inner container (ampoule) containing a nutrientmedium and an indicator for detecting biological activity, a firstsubstrate comprising a small circular metal coupon containingGeobacillus stearothermophilus spores coated thereon, a blue colored capfilter material (i.e., the “layer”, as described hereinabove), and aclosure. Additionally, a 1.27 cm diameter piece of DUPONT brand TYVEKmaterial was cut from a roll of 12407 TYVEK® Roll with STERRAD® ChemicalIndicator, available from Advanced Sterilization Products (ASP) ofIrvine, Calif. The circular cut piece of TYVEK material intentionallydid not include the chemical indicator, but was simply used as a secondsubstrate material on which the catalase was disposed. This TYVEK secondsubstrate was spiked with a 10 mg/mL aqueous solution of catalase, todeliver 200 μg of catalase per BI. The catalase spiked TYVEK materialwas allowed to dry. The modified Biological Indicator of Examples 1-5was made by reassembling the parts: first placing the spore-coated firstsubstrate into the bottom of the outer container; next the innercontainer containing the nutrient medium and the indicator for detectingbiological activity was placed into the outer container; and then thecatalase spiked second-substrate was placed into the outer container.Finally, the closure was friction-fitted over the opening of the outercontainer. Thus, in this Example, the blue colored cap filter material(i.e., the “layer”, as described herein) was replaced with the catalasespiked TYVEK material.

Example 2 was prepared in the same manner as Example 1 with theexception that a different second substrate material was used. Insteadof the TYVEK material cut from a roll of 12407 TYVEK® Roll with STERRAD®Chemical Indicator, a different non-woven web material was used, whichconsisted of 95/5 PP/rayon web material. The 95/5 PP/rayon material wasa carded non-woven web made of 95% polypropylene (single component)fibers and 5% LENZING (single component) rayon fibers at a basis weightof 135 gsm and carded through a two roll calendering method at 160° C.with one smooth roll and the other roll with an approximately 17% pointbond pattern. The rayon fibers were LENZING VISCOSE 1.7 dtex, 39 mm cutlength, bright raw white rayon fibers (Lenzing Fibers; New York, N.Y.The polypropylene fibers were FIBERVISIONS T-133/HY-Entangle 1.7 dtexpolypropylene fibers (FiberVisions Corporation; Duluth, Ga.).Additionally, Example 2 was spiked at 3 levels of catalase, to achieve:20 μg, 100 μg, and 200 μg of catalase per BI for Examples 2-1, 2-2 and2-3, respectively. The catalase spiked second substrate material wasallowed to dry and then the BIs were reassembled.

Example 3 was prepared in the same manner as Example 2, using the 95/5PP/Rayon web material, as the second substrate material. However, inExample 3 when the components of the 3M ATTEST 1274 BI were reassembled,after the spore-coated first substrate was placed into the bottom of theouter container; next the catalase spiked second substrate was placedover the spore-coated first substrate; then the inner containercontaining the nutrient medium and the indicator for detectingbiological activity was inserted, followed by the blue colored capfilter material. Finally, the closure was friction-fitted onto the outercontainer. Thus, in Example 3 the catalase was positioned in closerproximity to the spores, compared to Examples 1 and 2. Additionally,Example 3 was spiked at 2 levels of catalase, to achieve: 20 μg, and 200μg of catalase per BI for Examples 3-1, and 3-2, respectively. Thecatalase-spiked second substrate was allowed to dry and then the BIswere reassembled.

Example 4 was prepared in the same configuration as Example 3, in thatthe catalase-coated second substrate was positioned in between thespore-coated first substrate and the inner container containing thegrowth medium and indicator. However, Example 4 was prepared by using 3MATTEST 1292 Rapid Readout Biological Indicator; instead of the 3M ATTEST1274 BI. The 3M ATTEST 1292 Rapid Readout Biological Indicator has thesame number of components, oriented the same way, as the 1274 BI.However, the cap filter material (i.e., the “layer”, as described hereinabove) in the 3M ATTEST 1292 Rapid Readout BI is a white polypropylene(PP) meltblown web material. That PP web filter material was taken froma different 3M ATTEST 1292 Rapid Readout BI, and used as thecatalase-coated second substrate material. The aqueous catalase stocksolution was spiked onto this second substrate material to achieve aloading of about 200 μg of catalase per BI, allowed to dry and thenreassembled.

Example 5 was prepared by first disassembling several 3M ATTEST 1274Biological Indicators, then spiking aqueous catalase stock solutiondirectly onto the spores coated on the metal first substrate, allowed todry at room temperature conditions and then reassembling the biologicalindicators.

Reference Example 1

Reference Example 1, a control for Example 1 was prepared in exactly thesame manner as Example 1, except that the TYVEK second substratematerial was not spiked with catalase.

Reference Example 2

As with Reference Example 1, a control designated Reference Example 2,was prepared by preparing a sample exactly like Example 2, but with nocatalase spiked onto the 95/5 PP/Rayon web second substrate material.

Reference Example 3

As with Reference Examples 1 and 2, a control designated ReferenceExample 3, was prepared by preparing a sample exactly like Example 3,but with no catalase spiked on the 95/5 PP/Rayon web material.

Reference Example 4

Like the above Reference Examples, a control designated ReferenceExample 4 was prepared in exactly the same manner as Example 4, butwithout any catalase spiked on the second substrate material.

Reference Example 5

Like the above Reference Examples, a control designated ReferenceExample 5 was prepared in exactly the same manner as Example 5, butwithout any catalase spiked on the second substrate material.

TABLE 3 Location of Catalase as an Effect on Neutralization of HydrogenPeroxide in Biological Indicators Catalase Second Catalase- PositiveAmount Substrate coated Second BI/Total Example per BI MaterialSubstrate Location BI's Ref. Ex. 1 0 μg/BI TYVEK Near the Closure 0/10Ex.1 200 μg/BI TYVEK Near the Closure 0/10 Ref. Ex. 2 0 μg/BI PP/RayonNear the Closure 0/10 Ex. 2-1 20 μg/BI PP/Rayon Near the Closure 0/10Ex. 2-2 100 μg/BI PP/Rayon Near the Closure 0/10 Ex. 2-3 200 μg/BIPP/Rayon Near the Closure 0/10 Ref. Ex. 3 0 μg/BI PP/Rayon Above SporeCarrier 0/10 Ex. 3-1 20 μg/BI PP/Rayon Above Spore Carrier 0/10 Ex. 3-2200 μg/BI PP/Rayon Above Spore Carrier 6/10 Ref. Ex. 4 0 μg/BI PP WebAbove Spore Carrier 0/10 Ex. 4 200 μg/BI PP Web Above Spore Carrier10/10  Ref. Ex. 5 0 μg/BI None/Direct Directly on spores 0/12 Ex. 5 200μg/BI None/Direct Directly on spores 10/12 

Examples 6-9

The effect of different levels of various hydrogen peroxide-neutralizingagents on the resistance of spores to a hydrogen peroxide sterilizationprocess was evaluated in Examples 6-9. Commercially available 3M ATTEST1274 Biological Indicators were modified by the addition of variousneutralizing agents as described below. For each group of samples, 4similarly prepared biological indicators (BIs) were subjected to thesame sterilization cycle. Table 4 below shows the results of thesterilized biological indicators of Examples 6-9 as a ratio ofpositive/total BIs evaluated. A positive result implies that theneutralizing agent was effective in preventing the hydrogen peroxidesterilant from killing all the spores in the BI.

Example 6 was prepared by first disassembling several 3M ATTEST 1274Biological Indicators. Then, like Example 5, and amount of aqueouscatalase solution was spiked directly onto the spores on the metal sporecarrier coupon, to achieve 4 levels of catalase: 4 μg, 20 μg, 50 μg, and100 μg catalase per BI. The catalase solution was allowed to dry andthen the modified 3M ATTEST 1274 Biological Indicators were reassembledin their original configuration. Reference Example 6 was a control and,thus, had no catalase added.

Example 7 was prepared in the same manner as Example 6, except thatsodium thiosulfate was used instead of catalase as the hydrogenperoxide-neutralizing agent. The sodium thiosulfate, (Na₂S₂O₃)anhydrous, ≧98.0%, was obtained from Sigma Aldrich Company of St. Louis,Mo. Four levels of sodium thiosulfate were spiked onto the spores: 20μg, 100 μg, 200 μg and 2000 μg sodium thiosulfate per BI.

Example 8 was prepared in the same manner as Example 6, except thatsodium bisulfite was used instead of catalase as the hydrogenperoxide-neutralizing agent. The sodium bisulfite, (HNaO₃S) reagentgrade, was manufactured by BDH, and was obtained through VWR of Radnor,Pa. Four levels of sodium bisulfite were spiked onto the spores: 20 μg,200 μg, 250 μg and 2500 μg sodium bisulfite per BI.

Example 9 was prepared in the same manner as Example 6, except thatmethionine was used instead of catalase as the hydrogenperoxide-neutralizing agent. The methionine (99.00%) was obtained fromCalbiochem, EMD Chemicals of Gibbstown, N.J. and was spiked onto thespores at 500 μg per BI.

TABLE 4 Different Levels of Various Hydrogen Peroxide-NeutralizingAgents. The data show the number of growth-positive biologicalindicators for each group tested. With the exception of those designated“NT”, four biological indicators were tested per group. Concentration ofSTERRAD NX Neutralizing Agent 3 min. Half STERRAD NX OPTREOZ OPTREOZExample per BI Cycle Std Cycle H₂O₂ only Cycle 2 Ref. Ex. 6 0 (Control)0/4 0/4 0/4 0/4 Ex. 6-1 4 μg Catalase 0/4 0/4 0/4 0/4 Ex. 6-2 20 μgCatalase 4/4 0/4 4/4 0/4 Ex. 6-3 50 μg Catalase 4/4 4/4 4/4 4/4 Ex. 6-4100 μg Catalase 4/4 4/4 4/4 3/4 Ex. 7-1 20 μg Na₂S₂O₃ 0/4 0/4 4/4 0/4Ex. 7-2 100 μg Na₂S₂O₃ 4/4 2/4 4/4 0/4 Ex. 7-3 200 μg Na₂S₂O₃ 4/4 3/44/4 0/4 Ex. 7-4 2000 μg Na₂S₂O₃ 4/4 4/4 NT NT Ex. 8-1 20 μg HNaO₃S 2/40/4 0/4 3/4 Ex. 8-2 200 μg HNaO₃S 4/4 4/4 0/4 0/4 Ex. 8-3 250 μg HNaO₃S4/4 4/4 1/4 NT Ex. 8-4 2500 μg HNaO₃S 4/4 4/4 4/4 NT Ex. 9 500 μgMethionine 4/4 0/4 0/4 NT NT = Not tested

The complete disclosure of all patents, patent applications, andpublications, and electronically available material cited herein areincorporated by reference. In the event that any inconsistency existsbetween the disclosure of the present application and the disclosure(s)of any document incorporated herein by reference, the disclosure of thepresent application shall govern. The foregoing detailed description andexamples have been given for clarity of understanding only. Nounnecessary limitations are to be understood therefrom. The invention isnot limited to the exact details shown and described, for variationsobvious to one skilled in the art will be included within the inventiondefined by the claims.

All headings are for the convenience of the reader and should not beused to limit the meaning of the text that follows the heading, unlessso specified.

Various modifications may be made without departing from the spirit andscope of the invention. These and other embodiments are within the scopeof the following claims.

1-16. (canceled)
 17. An article, comprising: an outer container havingan interior volume, a first end comprising an opening, and a second end;a dry source of measurable biological activity disposed in the interiorvolume; and an effective amount of a dry agent for neutralizing asterilant compound, the effective amount being disposed in the interiorvolume; wherein both the source of measurable biological activity andthe agent are in vapor communication with an environment outside theouter container.
 18. The article of claim 17, further comprising a layerthat is permeable to the sterilant compound, wherein the layer iscoupled to the outer container, wherein the layer is interposed betweenthe source of measurable biological activity and the environment outsidethe outer container.
 19. The article of claim 17, wherein the source ofmeasurable biological activity comprises an enzyme activity or amicroorganism capable of reproducing.
 20. The article of claim 17,wherein the sterilant compound comprises hydrogen peroxide.
 21. Thearticle of claim 18, wherein the source of measurable biologicalactivity comprises an enzyme activity or a microorganism capable ofreproducing.
 22. The article of claim 18, wherein the sterilant compoundcomprises hydrogen peroxide.
 23. The article of claim 19, wherein thesterilant compound comprises hydrogen peroxide.